The present invention generally relates to materials and methods for production of D-amino acids and specifically D-phenylalanine.
With the exceptions of glycine, threonine, and isoleucine, each of the common, naturally-occurring amino acids exist as one of two optical isomers, termed levorotatory or dextrorotatory, depending upon the direction in which they cause the plane of plane-polarized light to rotate. Glycine, having no asymmetric carbon, has no optical isomers. Threonine and isoleucine, each having two asymmetric carbons, have four optical isomers each. Some amino acids, such as alanine and glutamine are dextrorotatory, producing a positive rotation. Others, such as phenylalanine and tryptophan, are levorotatory, producing a negative (left-handed) rotation. Thus, amino acids may be referred to as l- or d-amino acids in order to reflect their chirality in isolation.
The specific rotation produced by a given amino acid varies with temperature and pH. Accordingly, by convention, amino acids are also referred to as D or L (as opposed to the d or l designations referred to above) based upon whether the configuration about the xcex1-carbon of amino acid corresponds to the D or L stereoisomer (enantiomer) of glyceraldehyde, the arbitrary standard. Based upon that standard, most naturally-occurring amino acids are L-amino acids, despite the fact that certain of them are dextrorotatory (d) when placed in aqueous solution at neutral pH. Most enzymes which act upon amino acids have asymmetric binding domains which recognize only the L-form of the amino acid. Accordingly most naturally-occurring proteins comprise L-amino acids.
There are, however, exceptions wherein D-amino acids are produced and utilized by cells. Principal among these is the production of D-glutamate and D-alanine by certain microorganisms. D-glutamate and D-alanine are primarily produced in bacterial cells and are utilized in murein synthesis. In the absence of D-glutamate and D-alanine, a defective bacterial cell wall is produced, resulting in cell lysis. Most bacteria produce D-amino acids not by direct synthesis, but through conversion of the corresponding L-amino acid by an amino acid-specific racemase. For example, many bacterial cells possess an alanine racemase which catalyzes bidirectional conversion between L-alanine and D-alanine, resulting in a racemic (50:50) mixture. Similarly, a glutamate racemase produces a racemic mixture of D-glutamate and L-glutamate, the former for incorporation into the cell wall and the latter for, inter alia, formation of protein. The specificity of those two enzymes is demonstrated by the fact that the lack of either one results in cell lysis due to defective cell wall formation.
Certain bacteria, such as members of the genus Bacillus, possess an alternative to racemases for making D-amino acids in the form of D-aminotransferases. Such an enzyme reversibly catalyzes the transamination of various D-amino acids and corresponding xcex1-keto acids. In PCT Publication WO 91/05870, Manning reports a method for chemical synthesis of D-alanine and D-glutamate via catalysis by an aminotransferase. While Manning reports, at page 2, the use of a Bacillus sphaericus D-aminotransferase, that publication actually only reports the cloning, isolation, and use of a thermophilic species of D-aminotransferase which is not capable of effectively catalyzing synthesis of more than trace amounts of D-phenylalanine. Moreover, Manning fails to report any means for isolating or using a B. sphaericus D-aminotransferase or any other D-aminotransferase which catalyzes synthesis of D-phenylalanine.
Evidence that Manning""s reference to a B. sphaericus D-aminotransferase is an error is found at page 2 of the Manning publication, wherein Manning states that the D-aminotransferase DNA was cloned onto plasmid pICT113. As reported in Stoddard, et al., J. Mol. Biol., 196: 441-442 (1987), plasmid pICT113 carries the thermophilic species of D-aminotransferase and not the B. sphaericus species. The significance of that fact is that the thermophilic species cannot effectively catalyze significant production of D-phenylalanine and, therefore, is useless in recombinant methods for its production.
Prior to the present application, the only report of a B. sphaericus D-aminotransferase is a partial C-terminal sequence found in Transaminases, Christen, et al. (eds.), 464 (1985). However, as will be apparent from the present invention (See SEQ ID NO: 2), that partial sequence is wrong and is not useful in isolating the B. sphaericus D-aminotransferase. Accordingly, no prior reference reports a B. sphaericus D-aminotransferase gene sequence or the use of a B. sphaericus D-aminotransferase in the production, by recombinant means or otherwise, of a D-amino acid. Other D-aminotransferases have been isolated but, unlike the Bacillus sphaericus species, D-phenylalanine is a relatively poor substrate for those enzymes. Tanizawa, et al., J. Biol. Chem., 264: 2445-2449 (1989).
The present invention provides recombinant materials and methods for producing D-amino acids, including enantiomerically-pure D-amino acids, specifically D-phenylalanine.
The present invention provides methods for producing D-amino acids and recombinant microorganisms which produce D-amino acids. Microorganisms produced according to the invention contain a D-aminotransferase gene (dat) capable of effectively catalyzing production of D-phenylalanine in E. coli and optionally a dadX gene (encoding an alanine racemase), free of natural regulation, either integrated on a host chromosome or on a plasmid.
In a preferred embodiment of the invention, methods for producing D-phenylalanine are provided, said methods comprising the steps of incorporating into a microorganism a gene encoding a D-aminotransferase; increasing phenylpyruvic acid production; culturing the microorganism; and isolating D-phenylalanine produced thereby.
In a preferred embodiment, an alanine racemase, preferably a deregulated alanine racemase, and means for eliminating L-aminotransferases encoded by aspC, tyrB, and ilvE are also incorporated into the microorganism. The resulting inability to produce L-transaminases results in the production of entaniomerically-pure D-phenylalanine.
The invention also provides methods for the production of enantiomerically-pure D-phenylalanine; wherein a microorganism is transformed or transfected with a gene encoding a D-aminotransferase and optionally means for increasing phenylpyruvic acid production; and wherein L-aminotransferase activity is reduced or preferably eliminated.
The present invention provides microorganisms which produce D-phenylalanine. In a preferred embodiment, a microorganism of the invention is an Escherichia coli which has been transformed or transfected with an exogenous D-aminotransferase gene; an exogenous alanine racemase gene, such as a dadX gene of E. coli K12; and an exogenous aroH gene to increase throughput to the amino acid biosynthetic pathway via chorismate. An Escherichia coli of the invention also may comprise a pheA 34 gene as provided in co-owned U.S. Pat. No. 5,120,837, incorporated by reference herein, wherein the pheA coding sequence encodes a protein which is substantially resistant to feedback inhibition. Preferred microorganisms according to the invention were deposited on Mar. 22, 1995 with the American Type Culture Collection, 12310 Parklawn Drive, Rockville, Md. 20852 under ATCC accession numbers ATCC 69765 and ATCC 69766.
The present invention also provides a novel DNA encoding a B. sphaericus D-aminotransferase, plasmids containing that DNA, and host cells comprising the plasmids.
The present invention also provides methods for producing D-phenylalanine comprising transforming or transfecting an appropriate host cell with DNA encoding an aspartate racemase and with DNA encoding a phosphoenolpyruvate carboxykinase (a pckA gene), wherein the cell medium is supplemented with L-aspartic acid. Phosphoenolpyruvate carboxykinase catalyzes production of phosphoenolpyruvate from oxaloacetate. Finally, the present invention provides a preferred medium for culturing cells of the invention, said medium comprising from 2-7 g/L aspartic acid. Additional aspects of the invention are apparent to the skilled artisan upon consideration of the following detailed description.